PDB-9r5a: Crystal structure of an NtA622L variant in complex with NADP+

ID or keywords:

Entry

Database: PDB / ID: 9r5a

Title

Crystal structure of an NtA622L variant in complex with NADP+

Components

Isoflavone reductase homolog A622-like

Keywords

PLANT PROTEIN / Oxidoreductase / Nicotine / Niacin / NADPH / Rossmann-fold / IFR-like / PIP-reductase

Function / homology

Function and homology information

nicotine biosynthetic process / lignan biosynthetic process / oxidoreductase activity, acting on NAD(P)H, oxygen as acceptor / Oxidoreductases; Acting on the CH-CH group of donors; With NAD+ or NADP+ as acceptor / alkaloid metabolic process / secondary metabolite biosynthetic process / cytoplasm
Similarity search - Function

Biological species

Nicotiana tabacum (common tobacco)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.654 Å

Authors

Mokos, D. / Daniel, B.

Funding support

Austria, 3items

Citation

Journal: To Be Published
Title: A622: A key oxidoreductase in the Nicotine biosynthesis
Authors: Mokos, D. / Leitner, D. / Schruefer, A. / Grubmueller, S. / Reisz, C. / Juric, J. / Berger, T. / Singh, A. / Friess, M. / Breinbauer, R. / Gruber, K. / Daniel, B.

History

Deposition	May 8, 2025	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 20, 2026	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	9r5a.cif.gz	877.9 KB	Display	PDBx/mmCIF format
PDB format	pdb9r5a.ent.gz	602.6 KB	Display	PDB format
PDBx/mmJSON format	9r5a.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/r5/9r5a ftp://data.pdbj.org/pub/pdb/validation_reports/r5/9r5a	HTTPS FTP

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Related structure data

Related structure data	9qrgC 9qswC 9r1sC 9qwy C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Isoflavone reductase homolog A622-like

B: Isoflavone reductase homolog A622-like

C: Isoflavone reductase homolog A622-like

hetero molecules

106 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Isoflavone reductase homolog A622-like

hetero molecules

defined by author
35.4 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Isoflavone reductase homolog A622-like

hetero molecules

defined by author
35.4 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

3

C: Isoflavone reductase homolog A622-like

hetero molecules

defined by author
35.4 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	64.745, 65.241, 116.838
Angle α, β, γ (deg.)	90, 101.603, 90
Int Tables number	4
Space group name H-M	P12₁1

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details (eV)
1	1	A
2	1	A
3	2	A
4	2	A
5	3	A
6	3	A

NCS domain segments:

Auth asym-ID: A / Label asym-ID: A

Dom-ID	Component-ID	Ens-ID	Beg auth comp-ID	Beg label comp-ID	End auth comp-ID	End label comp-ID	Auth seq-ID	Label seq-ID
1	1	1	GLU	GLU	PHE	PHE	5 - 309	5 - 309
2	1	1	GLU	GLU	PHE	PHE	5 - 309	5 - 309
3	2	2	SER	SER	VAL	VAL	4 - 310	4 - 310
4	2	2	SER	SER	VAL	VAL	4 - 310	4 - 310
5	3	3	GLU	GLU	PHE	PHE	5 - 309	5 - 309
6	3	3	GLU	GLU	PHE	PHE	5 - 309	5 - 309

NCS ensembles :

ID	Details (eV)
1	Local NCS retraints between domains: 1 2
2	Local NCS retraints between domains: 3 4
3	Local NCS retraints between domains: 5 6

#1: Protein	Isoflavone reductase homolog A622-like / NtA622L Mass: 34640.734 Da / Num. of mol.: 3 / Mutation: K99N M201I M259L Source method: isolated from a genetically manipulated source Source: (gene. exp.) Nicotiana tabacum (common tobacco) / Gene: A622L, IRL2 / Production host: Escherichia coli (E. coli) References: UniProt: B6VRE6, Oxidoreductases; Acting on the CH-CH group of donors; With NAD+ or NADP+ as acceptor
#2: Chemical	ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE Mass: 743.405 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C₂₁H₂₈N₇O₁₇P₃ / Feature type: SUBJECT OF INVESTIGATION
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 540 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	Y
Has protein modification	N

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.33 Å³/Da / Density % sol: 47.12 %
Crystal grow	Temperature: 291 K / Method: vapor diffusion, sitting drop / pH: 5.5 Details: Index screen condition 78: 0.1 M Ammonium acetate, 0.05 M BIS-TRIS pH 5.5, 12.5% w/v Polyethylene glycol 3,350; 5mg/ml protein, 1mM NADP+, 1mM Trigonelline

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

ESRF

/ Beamline: MASSIF-3 / Wavelength: 0.9677 Å

Detector

Type: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Oct 27, 2024

Radiation

Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.9677 Å / Relative weight: 1

Reflection

Resolution: 1.65→47.504 Å / Num. obs: 112073 / % possible obs: 98.4 % / Redundancy: 3.5 % / CC_1/2: 0.996 / R_merge(I) obs: 0.074 / R_pim(I) all: 0.068 / R_rim(I) all: 0.101 / Net I/σ(I): 8

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Num. unique obs	CC_1/2	R_pim(I) all	R_rim(I) all
9.06-47.5	2.9	0.05	712	0.978	0.049	0.07
1.65-1.68	2.6	0.984	4819	0.244	0.91	1.344

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0425	refinement
Aimless		data scaling
XDS		data reduction
Coot		model building
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.654→47.504 Å / Cor.coef. F_o:F_c: 0.962 / Cor.coef. F_o:F_c free: 0.949 / SU B: 5.677 / SU ML: 0.09 / Cross valid method: FREE R-VALUE / ESU R: 0.106 / ESU R Free: 0.101
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.2185	5570	4.971 %
R_work	0.1911	106480	-
all	0.192	-	-
obs	-	112050	98.289 %

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT

Displacement parameters

B_iso mean: 32.746 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.131 Å²	0 Å²	-0.033 Å²
2-	-	0.378 Å²	-0 Å²
3-	-	-	-0.216 Å²

Refinement step

Cycle: LAST / Resolution: 1.654→47.504 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	7263	0	144	540	7947

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.011	0.012	7611
X-RAY DIFFRACTION	r_bond_other_d	0.004	0.016	7289
X-RAY DIFFRACTION	r_angle_refined_deg	2.076	1.827	10364
X-RAY DIFFRACTION	r_angle_other_deg	1.01	1.752	16854
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.739	5	923
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	5.902	5	27
X-RAY DIFFRACTION	r_dihedral_angle_other_2_deg	12.808	5	6
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.554	10	1309
X-RAY DIFFRACTION	r_dihedral_angle_6_deg	15.861	10	320
X-RAY DIFFRACTION	r_chiral_restr	0.135	0.2	1193
X-RAY DIFFRACTION	r_gen_planes_refined	0.011	0.02	8588
X-RAY DIFFRACTION	r_gen_planes_other	0.005	0.02	1623
X-RAY DIFFRACTION	r_nbd_refined	0.187	0.2	1308
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.134	0.2	6424
X-RAY DIFFRACTION	r_nbtor_refined	0.157	0.2	3678
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.062	0.2	4061
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.118	0.2	451
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.016	0.2	1
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.135	0.2	20
X-RAY DIFFRACTION	r_nbd_other	0.147	0.2	83
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.088	0.2	14
X-RAY DIFFRACTION	r_mcbond_it	1.842	1.664	3692
X-RAY DIFFRACTION	r_mcbond_other	1.842	1.664	3692
X-RAY DIFFRACTION	r_mcangle_it	2.704	2.993	4615
X-RAY DIFFRACTION	r_mcangle_other	2.704	2.994	4616
X-RAY DIFFRACTION	r_scbond_it	2.717	1.958	3919
X-RAY DIFFRACTION	r_scbond_other	2.717	1.958	3917
X-RAY DIFFRACTION	r_scangle_it	4.211	3.462	5749
X-RAY DIFFRACTION	r_scangle_other	4.211	3.462	5750
X-RAY DIFFRACTION	r_lrange_it	6.402	17.812	8372
X-RAY DIFFRACTION	r_lrange_other	6.36	17.546	8258
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.098	0.05	9738
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.089	0.05	9907
X-RAY DIFFRACTION	r_ncsr_local_group_3	0.091	0.05	9783

Refine LS restraints NCS

Ens-ID	Dom-ID	Auth asym-ID	Refine-ID	Type	Rms dev position (Å)	Weight position
1	1	A	X-RAY DIFFRACTION	Local ncs	0.09765	0.05007
1	2	A	X-RAY DIFFRACTION	Local ncs	0.09765	0.05007
2	3	A	X-RAY DIFFRACTION	Local ncs	0.08862	0.05007
2	4	A	X-RAY DIFFRACTION	Local ncs	0.08862	0.05007
3	5	A	X-RAY DIFFRACTION	Local ncs	0.09119	0.05007
3	6	A	X-RAY DIFFRACTION	Local ncs	0.09119	0.05007

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	R_factor all	Num. reflection all	Fsc free	Fsc work	% reflection obs (%)	WR_factor R_work
1.654-1.697	0.35	347	0.34	7115	0.341	8384	0.901	0.906	89.0029	0.338
1.697-1.743	0.343	409	0.324	7659	0.325	8158	0.914	0.928	98.8968	0.317
1.743-1.794	0.329	383	0.295	7544	0.296	7939	0.932	0.947	99.8488	0.28
1.794-1.849	0.296	379	0.261	7334	0.262	7720	0.951	0.959	99.9093	0.241
1.849-1.91	0.264	357	0.233	7170	0.235	7539	0.955	0.966	99.8408	0.213
1.91-1.976	0.244	328	0.224	6856	0.225	7196	0.962	0.969	99.8332	0.202
1.976-2.051	0.243	347	0.207	6630	0.208	6989	0.966	0.974	99.8283	0.186
2.051-2.135	0.233	323	0.205	6415	0.207	6755	0.966	0.974	99.7483	0.187
2.135-2.229	0.197	352	0.184	6100	0.185	6478	0.975	0.98	99.5986	0.169
2.229-2.338	0.221	318	0.179	5805	0.181	6172	0.971	0.981	99.2061	0.164
2.338-2.464	0.205	289	0.171	5493	0.173	5848	0.974	0.982	98.8714	0.16
2.464-2.613	0.199	240	0.177	5267	0.178	5586	0.973	0.981	98.5857	0.168
2.613-2.793	0.214	274	0.178	4836	0.18	5267	0.971	0.98	97.0192	0.172
2.793-3.016	0.216	228	0.186	4379	0.188	4842	0.969	0.979	95.1466	0.185
3.016-3.302	0.219	232	0.196	4185	0.198	4542	0.97	0.978	97.2479	0.199
3.302-3.69	0.208	254	0.188	3793	0.189	4072	0.978	0.981	99.386	0.194
3.69-4.256	0.182	162	0.153	3440	0.154	3628	0.98	0.986	99.2833	0.165
4.256-5.202	0.168	183	0.136	2860	0.138	3064	0.987	0.991	99.3146	0.152
5.202-7.313	0.176	118	0.157	2296	0.158	2424	0.983	0.988	99.5875	0.171
7.313-47.504	0.263	47	0.187	1303	0.189	1392	0.978	0.977	96.9828	0.198

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°²)	L12 (°²)	L13 (°²)	L22 (°²)	L23 (°²)	L33 (°²)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å²)	T12 (Å²)	T13 (Å²)	T22 (Å²)	T23 (Å²)	T33 (Å²)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	0.6565	-0.15	0.0278	0.9399	-0.4441	0.6136	0.0557	-0.077	0.0508	-0.0522	0.0254	-0.0036	0.054	-0.0246	-0.0811	0.0964	-0.0082	0.0174	0.0594	-0.016	0.0956	5.5962	-0.6451	17.4046
2	0.6439	0.0811	-0.0958	1.6608	0.1737	0.7403	-0.0134	0.0993	0.0247	-0.024	0.0588	-0.0701	-0.035	0.0622	-0.0454	0.0248	0.0214	0.0412	0.1099	0.0286	0.0957	26.1388	-2.6433	-19.7853
3	0.3417	-0.0595	0.0264	1.1391	-0.7289	1.9181	-0.0447	-0.2553	-0.0741	0.1421	0.0558	-0.1157	-0.1102	-0.5018	-0.0111	0.0526	0.0396	-0.0092	0.4187	0.0433	0.0564	3.7581	-16.4698	55.59

Refinement TLS group

Selection: ALL

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